Vitamin B12 and Folate Markers Are Associated with Insulin Resistance During the Third Trimester of Pregnancy in South Asian Women, Living in the United Kingdom, with Gestational Diabetes and Normal Glucose Tolerance

ABSTRACT Background Gestational diabetes mellitus (GDM) can adversely affect the health of the developing fetus. Women of South Asian origin are particularly at risk of developing GDM. Insulin resistance (IR) contributes to the etiology of GDM, and although studies have shown associations of vitamin B12 (B12) and folate status with GDM and IR, only a limited number of B12 and folate markers have been used. Objective We used a comprehensive panel of B12 and folate markers to examine their association with IR in pregnant women with diet-controlled GDM and normal glucose tolerance (NGT). Methods In this cross-sectional study, 59 British-Bangladeshi women (24 GDM and 35 NGT) with a mean age of 29 y, BMI (in kg/m2) 26.7 and gestational age 33 wk were recruited. Serum total B12, holotranscobalamin, folate, methylmalonic acid, plasma homocysteine, 5-methyltetrahydrofolate, and red cell folate (RCF) were measured along with other parameters. The independent sample t-test and chi-squared test were used to assess differences in markers between GDM and NGT women. Spearman's test was used to look for correlations. A simple multiple regression analysis was used to investigate if markers of B12 and folate status predicted IR, using the HOMA-IR and adjusting for age, GDM status, and BMI. Results There were no differences in concentrations of B12 and folate markers between GDM and NGT women. In Spearman's analysis HOMA-IR correlated negatively with total serum B12 (P < 0.001) and holotranscobalamin (P < 0.05), and positively with BMI (P < 0.001), blood pressure (P < 0.05) and triglycerides (P < 0.05) in all women. MMA did not correlate with any of the B12 markers. In regression analysis, total B12 (β = −0.622, P = 0.004), RCF (β = 0.387, P = 0.018), and BMI (β = 0.024, P < 0.001) were the significant predictors of HOMA-IR variance. Conclusions Significant associations between markers of B12 and folate status with HOMA-IR were found during the third trimester in British-Bangladeshi women. B12 markers correlated poorly with each other.


Introduction
A healthy maternal environment is critical to fetal development. Gestational diabetes mellitus (GDM) (glucose intolerance in pregnancy) is one of the most common disorders of pregnancy. Insulin resistance (IR) is thought to contribute to the etiology of GDM. Pregnancy naturally predisposes to IR as a result of the physiological adaptation necessary to provide glucose to the growing fetus. In early pregnancy reduced IR promotes maternal adipose tissue accumulation. As pregnancy progresses increased maternal IR helps nutrient transfer to promote fetal growth (1). To compensate for IR, there is increased production of insulin from pancreatic beta cells. In normal pregnancies, there is an approximate 50% decrease in insulin-mediated glucose disposal in late pregnancy and a 200% to 250% C The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Manuscript received April 9, 2021. Initial review completed June 21, 2021. Revision accepted September 24, 2021. First published online November 9, 2021; doi: https://doi.org/10.1093/jn/nxab352. increase in insulin secretion to maintain euglycemia in the mother (2). GDM results from a reduced capacity for insulin production, and it has been suggested that women who develop GDM have pre-existing defects in insulin action and secretion and are, therefore, predisposed to developing type 2 diabetes (3,4).
A family history of diabetes, BMI (in kg/m 2 ) >30, previous GDM, a previous macrosomic baby weighing ≥4.5 kg, and ethnicity are common clinical risk factors for developing GDM. Women of South Asian origin are particularly at risk of developing GDM due to genetic, intrauterine, socioeconomic, and behavioral risk factors. In some Asian countries the prevalence of GDM reaches 25% compared with ∼10% in North America (5,6). GDM is also associated with adverse effects on the fetus, e.g., macrosomia, neonatal hypoglycemia, and long-term risk of adiposity and type 2 diabetes via fetal programming (7)(8)(9)(10)(11).
Lower muscle mass, higher percentage of body fat and higher IR compared with other populations are the characteristic phenotypic features which have been observed in Asians, as early as at birth (12). Intrauterine undernutrition during pregnancy was suggested as a contributing factor to this phenotype (12). A study conducted in India found that higher maternal folate concentrations during pregnancy predicted greater adiposity and higher IR in the offspring, and lower vitamin B 12 (B 12 ) concentrations predicted higher IR (13). Children born to mothers with high folate but low B 12 concentrations were the most insulin resistant (13). Other studies have also suggested that excessive use of folic acid (commonly taken as a pregnancy supplement) may adversely affect the health of the fetus, especially in combination with low B 12 , and that this may be additionally associated with GDM risk and IR (13)(14)(15)(16)(17). The mechanisms behind this relation are currently unknown but could include a functional folate deficiency through the methyltrap phenomenon, leading to DNA hypomethylation, impaired metabolism of homocysteine causing endothelial dysfunction, and oxidative stress (5). Deficiency of B 12 also impacts transfer of fatty acids into mitochondria, resulting in impaired betaoxidation. This would promote lipo-and adipogenesis and consequently increased risk of IR (5,18).
To date, B 12 and folate status in the studies linking IR and GDM with B 12 and folate status have predominately been assessed using serum total B 12 or folate measurements while applying non-pregnancy derived cutoffs for the interpretation of these markers. Serum total B 12 in particular has been criticized as a marker owing to its lack of sensitivity to B 12 deficiency (19), and it is difficult to interpret this marker because serum B 12 concentrations decrease during pregnancy because of hemodilution and decreased synthesis of haptocorrin (the most abundant B 12 binding protein). The functional markers of B 12 deficiency, serum methylmalonic acid (MMA) and total plasma homocysteine (Hcy), are also affected in pregnancy (20).
The primary aim of this work was to evaluate associations of B 12 and folate status with IR using a comprehensive panel of markers during the third trimester of pregnancy in British-Bangladeshi women with normal glucose tolerance (NGT) and GDM. To evaluate B 12 status we used serum total B 12 , serum HoloTC, serum MMA, plasma Hcy, and a combined indicator of B 12 status (cB 12 ); and for folate status we used serum and red cell folate (RCF) and plasma 5-methyltetrahydrofolate (5-MTHF). We also set out to evaluate B 12 and folate status using the typical non-pregnancy related cutoffs which are routinely applied to pregnant women in the UK. This investigation forms a secondary analysis in a study designed to investigate fetal epigenetic differences associated with maternal gestational diabetes (8).

Subjects
Pregnant British-Bangladeshi women with and without GDM, attending the antenatal care unit at the Royal London Hospital, London, United Kingdom, were recruited during the third trimester of pregnancy (8). The diagnosis of GDM was made based on a 2-h 75-g oral glucose tolerance test (OGTT) following an 8-h fast, using a standard clinical protocol, as part of routine antenatal care at 28 wk of gestation. Blood sampling was taken at 0 and 120 min post glucose ingestion. Local diagnostic cutoffs were used for GDM diagnosis (0-min serum glucose ≥5.8 and/or 120-min ≥7.8 mmol/L). Women with multiple pregnancies or pre-existing diabetes, as well as women who were taking metformin and/or were on insulin therapy, were excluded from the analysis.
At recruitment, the women were asked about iron, folic acid, and multivitamin usage during pregnancy, and based on their responses we grouped these subjects into iron, folic acid, or multivitamin users.
Blood samples were taken to assess folate and B 12 status, total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides (TG). Blood pressure (BP) was also recorded.
Pregnancy notes were used to collect anthropometric data in the first trimester. Pregnancy history was recorded. Neonatal measurements included the following: weight, height, head circumference (HC), chest circumference (CC), and abdominal circumference. The study was approved by the NHS Research Ethics Committee (Ref 09/H0718/31; IRAS ID 10031).

Analytical methods
Fasting blood samples (30 ml) following an 8-h fast were collected from each woman after a diagnosis toward GDM had been made and standard dietary management had been initiated (22). Blood samples were placed on ice, and whole blood was separated into serum/plasma within 2 h of collection using standard procedures. Serum/plasma samples were stored at −80 • C until analysis. Fasting insulin was measured by an electrochemiluminescent sandwich immunoassay (Roche) and serum glucose (as part of an oral glucose tolerance test) by hexokinase assay (Roche).
Serum cholesterol, TG, and LDL-, and HDL cholesterol were measured on Cobas 6000 (Roche) with satisfactory analytical performance monitored through use of the UKNEQAS, external quality assurance (EQA) scheme.
Serum folate and total serum B 12 were performed by an electrochemiluminescent competitive immunoassay (Roche), whereas a competitive binding paramagnetic particle assay (Beckman Coulter) was used for RCF. These tests were performed by Barth Health NHS Trust, London, United Kingdom, with satisfactory analytical performance monitored through use of the UKNEQAS EQA scheme. We applied the standard reference ranges used at Barts Health NHS for these tests as these are routinely applied to all subjects, including pregnant women. The reference ranges are: 8.6-45.3 nmol/L for serum folate, 140-664 pmol/L for serum total B 12 , and 362-1450 nmol/L for RCF. Serum Hcy was measured by HPLC at Homerton NHS Trust, EQA scheme UKNEQAS; cutoff for pregnancy <10 μmol/L. Serum HoloTC and MMA and plasma 5-MTHF were measured at the Nutristasis Unit, Viapath, St. Thomas' Hospital. Serum HoloTC analysis was carried out using an Architect 2000 (Abbott Diagnostics). For 5-MTHF, an HPLC assay was utilized as described elsewhere (20,23). LC-MS/MS was used for the determination of MMA (Gerstel Multi-Purpose Sampler coupled directly to Agilent Technologies 6460 Triple Quad) (24). External quality assurance schemes were used for all assays; UKNEQAS for HoloTC and 5-MTHF and DEKS for MMA. Vitamin B 12 deficiency is suggested if HoloTC is <25 pmol/L and MMA >280 nmol/L, whereas folate deficiency is suggested if plasma 5-MTHF is <7.6 nmol/L (23,25).
HOMA-IR, which is derived from a mathematical assessment of the balance between hepatic glucose output and insulin secretion, was calculated using fasting glucose and insulin concentrations (27,28). Increased values of HOMA-IR are indicative of IR.

Statistical analysis
The Shapiro-Wilk test was used to check the normality of data. Serum total B 12 , HoloTC, and folate and plasma 5-MTHF and RCF results were inspected for outliers using the outlying labeling rule, g = 2.2 (29), to identify women on potentially very high B 12 or folate supplements. As a result of these analyses, 1 serum B 12 , 4 HoloTC, 1 RCF, and 1 5-MTHF result were excluded from the analyses. Where applicable, results not normally distributed were log transformed before analysis. We used descriptive statistics to summarize characteristics of women with NGT and GDM, including their B 12 and folate status, and applied the Wilcoxon signed rank test, independent sample t-test, and chi-squares tests (as appropriate) to test for differences between women with NGT and with GDM, and the selected baby parameters from babies born to NGT and GDM mothers. Spearman's test was used to investigate correlations, first between HOMA-IR with other variables measured and second between B 12 and folate markers. Multiple regression analyses was used to study independent predictors of HOMA-IR from the selected variables: age, gestational age, GDM status, BMI, diastolic pressure, serum total B 12 , serum HoloTC, serum MMA, plasma Hcy, RCF, serum folate, and plasma 5-MTHF. Due to multicollinearity between B 12 and folate markers, separate models were constructed by adding serum B 12 and HoloTC and serum folate, 5-MTHF, RCF, and Hcy separately. Statistical analyses were carried out using SPSS, v. 26.

Characteristics of the study population
Compared with women without GDM, women with GDM were older (P = 0.035), had a higher gestational age at the time of testing (P = 0.035), and had higher glucose concentrations during OGTT ( Table 1). No other measurements related to adiposity or diabetic predisposition differed between the 2 groups (Table 1).

Vitamin status and the prevalence of deficiencies/high status based on routinely-used cutoffs
There were no differences in the concentrations of B 12 and folate markers between NGT and GDM women ( Table 2). The numbers of women taking iron supplements and folic acid were similar in the 2 groups but there was an excess of multivitamin supplement users in the GDM group (P = 0.039). Combined analysis of the whole group (n = 59) ( Table 2) showed variations in estimation of the prevalence of B 12 deficiency when using individual markers and applying routinely used nonpregnant reference ranges for most markers: 21% with serum total B 12 (<140 pmol/L) , 3% with HoloTC (<25 pmol/L), 5% with serum Hcy (>10 μmol/L; suggested for pregnancy), and 31% with serum MMA (>280 nmol/L) (25,30). Only 5% of women had folate deficiency as estimated by serum folate (<8.6 nmol/L) or RCF (<362 nmol/L), whereas 14% of women had low plasma 5-MTHF (<7.6 nmol/L) (23,31). Five percent of women had high serum folate (>45.3 nmol/L), 7% had high RCF (>1450 nmol/L) and 20% had high plasma 5-MTHF (>42.0 nmol/L) (32). There was no difference in the prevalence of B 12 and folate deficiency or high folate concentrations between NGT and GDM women (data not shown).
Examining the association of comprehensive B12 and folate markers with HOMA-IR Bivariate correlations (Spearman's test) of fasting glucose and fasting insulin with B 12 and folate markers showed that fasting glucose correlated negatively with HoloTC alone (ρ = −0.272, P = 0.037), and fasting insulin correlated negatively with serum total B 12 (ρ = −0.402, P = 0.002) and HoloTC (ρ = −0.306, P = 0.020) but not with MMA, cB12, Hcy, and folate markers. HOMA-IR correlated negatively with serum total B 12 and HoloTC and positively with BMI, BP, and TG in all women ( Table 3). In a multiple regression analysis, serum total B 12 , RCF, and BMI were the only significant predictors of HOMA-IR variance ( Table 4).
In another multiple regression model with HoloTC as a variable instead of serum total B 12 , HoloTC did not have a statistically significant association with HOMA-IR (P = 0.197). Serum folate (P = 0.352), plasma 5-MTHF (P = 0.174), and Hcy (P = 0.145) were also not significant predictors of HOMA-IR when added separately to the models (data not shown). Lipids were not included in multiple regressions models because of high percentages of missing data.

Bivariate correlations between markers of B 12 and folate status
Since our bivariate correlations and multiple regression analysis showed consistently negative associations between serum total B 12 (and HoloTC in the correlation) and HOMA-IR, but the same was not seen for MMA and Hcy, bivariate correlations between all B 12 markers were carried out to see if the expected relations between these markers were present ( Table 5).

Discussion
We found significant associations between serum total B 12 , HoloTC, and RCF with IR (using the homeostatic model HOMA-IR) in Bangladeshi women during the third trimester of pregnancy. These findings were consistent across bivariate analyses (negative associations for serum total B 12 and HoloTC) and multivariate analyses (negative associations for serum total B 12 and positive for RCF). These findings are in agreement with those of other studies which have also reported that lower serum total B 12 concentrations are associated with a higher HOMA-IR index during the third trimester of pregnancy (14,33). Unlike previously reported studies which have shown associations between GDM risk and B 12 and folate status using total serum B 12 and folate tests, we used a comprehensive panel of biochemical markers of B 12 and folate status. Most previous studies had quantified serum total B 12 only, and it is known that this test in particular is greatly affected by pregnancy because of hormonal and B 12 binding protein changes (34,35) in addition to declining B 12 stores due to transfer to the fetus (20). These factors reduce the diagnostic utility of total B 12 during pregnancy. Our correlation and regression analyses showed that both serum total B 12 and HoloTC were negatively associated with HOMA-IR. These results confirm previously reported associations and suggest a possible role for B 12 in IR/GDM risk. It was also of interest to see the significant variations in the prevalence of B 12 deficiency, in particular when serum total B 12 and HoloTC markers only were considered, 21% compared with 3%, respectively, and this indicates that the routinelyused cutoffs that are applied to pregnant women are unhelpful. Previous pregnancy studies suggested that HoloTC may be a better marker of B 12 status during pregnancy than total B 12 as it is less affected by hormonal changes and by the decrease in the levels of haptocorrin during pregnancy (21,36). Interestingly, Fernandez-Costa and Metz (37) reported a sharp increase in HoloTC during the third trimester, whereas Murphy et al. (36) found that after the initial reduction in plasma HoloTC in early pregnancy, HoloTC reached a plateau and remained at a lower level until the end of pregnancy. The authors suggested that compensatory mechanisms operate during pregnancy to ensure a B 12 supply sufficient to meet increased B 12 requirements in both the mother and the fetus (36). Thus, use of total serum B 12 may overestimate prevalence of B 12 deficiency in pregnancy. Consequently, HoloTC could be a better diagnostic tool for the assessment of B 12 status during the third trimester of pregnancy than serum total B 12, as supported by our observation that HoloTC levels correlate with homocysteine in our study.
No association of HOMA-IR with MMA was found, and this is an important finding as this test is often used for confirmation of B 12 status assessment (25,38). The prevalence of elevated MMA was 31%, but the usual correlations of MMA with serum total B 12 , HoloTC, and Hcy were not seen in this study. Interestingly, only 3 (17%) of the women with MMA >280 nmol/L had serum total B 12 <140 pmol/L. This finding could suggest that not only serum total B 12 but also MMA concentrations may be influenced by factors not related to B 12 status during the third trimester of pregnancy. Hemodilution and hormonal changes have been likewise proposed as modulators of MMA concentration during pregnancy (36). Methylmalonic acid is a product of mitochondrial hydrolysis of excess methylmalonyl-CoA (MMA-CoA), which is formed from the conversion of odd-chain fatty acids, cholesterol and branched chain amino acids, via propionate. MMA-CoA is converted to succinyl-CoA, a major intermediate of the citric acid cycle, by a mutase with B 12 serving as a cofactor. The inhibition of this pathway in B 12 deficiency leads to the accumulation of MMA-CoA, which can inhibit carnitine palmitoyl transferase system, an essential step in the betaoxidation of long fatty acids, thus promoting lipogenesis and insulin resistance (14,39,40). Conversely, the higher use of fats than carbohydrates for energy toward the end of pregnancy may also result in more MMA-CoA being converted to MMA regardless of B 12 status.
Similarly to MMA, another functional marker of B 12 deficiency, Hcy, did not show any associations with HOMA-IR and did not correlate with serum total B 12 nor MMA, but it correlated with HoloTC. Increased Hcy concentrations and their impact on the impairment of endothelial function leading to diabetes (16) have been proposed as another mechanism contributing to GDM risk. However, whilst some studies demonstrated differences in Hcy concentrations between women with NGT and GDM or associations with GDM risk (16) (41)(42)(43), others failed to observe the same (17,44). This is possibly because Hcy concentrations are normally much lower during pregnancy. Hemodilution, hormonal changes, and folic acid supplementation are probably the main reasons for low concentrations of Hcy in pregnancy. Moreover, subnormal Hcy was reported in nondiabetic hyperinsulinemic subjects in the prediabetic stage (45). Glomerular hyperfiltration observed in early diabetes and metabolic effects of high insulin levels have been proposed as mechanisms for low Hcy (46,47). Therefore, Hcy concentrations during the third trimester may be significantly dependent on metabolic and hormonal changes and thus may not be reliable as a marker of B 12 or folate status. More evidence is required to demonstrate that higher homocysteine may be associated with higher IR and GDM risk.
cB12 estimation provides a more comprehensive evaluation of B 12 status as it takes into account all B 12 markers and makes adjustments for patient age and folate status. So far, cB12 has not been validated in pregnancy. As serum total B 12 , MMA and Hcy in particular are probably affected by other factors independent of B 12 status during pregnancy, the utility of cB12 as a marker in pregnancy needs further evaluation. As expected, cB12 correlated with all B 12 markers. The prevalence of low B 12 status assessed using cB12 was similar to that assessed using HoloTC.
The underlying mechanisms for the associations of HOMA-IR with B 12 status during the third trimester of pregnancy are currently unknown. Although the role of B 12 in lipid metabolism has not been fully elucidated, the inhibition of fatty acid oxidation is one of the proposed mechanisms leading to insulin resistance (13). Studies have demonstrated that adipocytes cultured in low B 12 conditions had increased cholesterol and homocysteine concentrations combined with hypomethylated promoter regions of the key regulator genes (SREBF1 and the LDR receptor (LDLR)) of cholesterol biosynthesis (48) as well as a significantly increased expression of genes involved in triglyceride biosynthesis and decreased expression of βoxidation genes (49). Other in vitro studies have found that adipocytes in low B 12 conditions displayed increased lipid accumulation (50). In our study, triglycerides correlated positively with HOMA-IR (Table 3) and negatively with serum total B 12 (ρ = −0.345, P = 0.034) (data not shown). Thus, maternal body fat distribution/metabolism during pregnancy may be implicated in determining circulating concentrations of micronutrients in pregnancy as previously suggested (33). As BMI is a strong predictor of HOMA-IR, it will be important for future studies to elucidate whether body fat has a causal role in the relation between B 12 status and HOMA-IR, or whether this is confounded by other factors such as socioeconomic status and vitamin supplement use. Such an analysis is not possible in a cross-sectional study.
No significant differences were found between women with NGT and GDM with regard to B 12 status. This was true for all B 12 markers used, including cB12. This is unlike the results reported by Krishnaveni et al. (14) and Sukumar et al. (15), who identified higher rates of B 12 deficiency in pregnant women with GDM than those with NGT in South Indian and white European populations, respectively. Results of a recent systematic review and meta-analysis also suggested that pregnant women with B 12 deficiency are at almost 2-fold higher risk of developing GDM than women with B 12 sufficiency (51). However, it should be noted that this meta-analysis included only 2 studies (14,15), which are discussed above. The findings in our study may have been influenced by the small number of participants, and because of this we included women who were taking supplements. However, other studies do concur with our finding that there is no difference in B 12 status between women with NGT and GDM (17,44,52). Future studies examining this relation should examine the direct effects of GDM management on B 12 and folate status. Current dietary recommendations predominantly focus on carbohydrate restriction and it is conceivable that a reduction in carbohydrate intake could result in replacement by foods which have a greater B 12 and/or folate content. Although no data were collected showing the supplementation/medication changes after GDM diagnosis, the finding of 4 women with GDM with highly elevated HoloTC (extreme outliers) as well as the higher proportion of women with GDM taking vitamin supplements may support this notion. Moreover, unlike total serum B 12 , HoloTC reflects a more recent intake of B 12 , and GDM diagnosis preceded the sample collection for this study.
In contrary to B 12 markers, folate markers utilized in this study correlated highly with each other: (ρ ≥ 0.8, P < 0.001). The prevalence of low and high folate status was higher when 5-MTHF was used as a marker of folate assessment than when serum folate and RCF were used. Methodological differences and cutoff variations, as well as small sample, size may partially account for this finding. The study size did not permit the partition by folate status subgroups incorporating low/normal B 12 concentrations which could be used to establish if women with low B 12 and high folate status were more prevalent in the GDM group.
Importantly, RCF was a significant positive predictor of HOMA-IR variance (Table 4). Serum folate and plasma 5-MTHF, included in separate models, did not reach the statistical significance in predicting HOMA-IR variance. RCF is a marker of long-term folate status, whereas serum folate is affected by recent folate intakes (31). Diet control could have had an impact on both serum folate and plasma 5-MTHF. Both low and high serum folate concentrations have been associated with negative health effects (32,53), and it has been suggested that folate may affect the development of GDM via different pathways (16). More work is needed to address optimum and safe folate supplementation regimes during pregnancy.
The strong associations of HOMA-IR with BMI were also confirmed in this study, both in multivariate and bivariate analysis, as well as the previously reported associations of IR with infant length (54,55).

Conclusions
In conclusion, B 12 and folate status are associated with IR during the third trimester of pregnancy in South Asian women living in the United Kingdom. As the provision of balanced and adequate B 12 and folate is vital for the wellbeing of both mother and child, markers of status need to be carefully interpreted in view of pregnancy-related changes affecting concentrations of these nutrients. Pregnancy-specific reference ranges should be used for B 12 markers. Replication of these findings in larger cohorts may provide valuable insights into the mechanisms linking B 12 and folate status with IR and GDM.